Fontes Joseph D, Ramsey Jon, Polk Jeremy M, Koop Andre, Denisova Janna V, Belousov Andrei B
Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, Kansas, United States of America.
Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, Kansas, United States of America.
PLoS One. 2015 May 27;10(5):e0125395. doi: 10.1371/journal.pone.0125395. eCollection 2015.
Pharmacological blockade or genetic knockout of neuronal connexin 36 (Cx36)-containing gap junctions reduces neuronal death caused by ischemia, traumatic brain injury and NMDA receptor (NMDAR)-mediated excitotoxicity. However, whether Cx36 gap junctions contribute to neuronal death via channel-dependent or channel-independent mechanism remains an open question. To address this, we manipulated connexin protein expression via lentiviral transduction of mouse neuronal cortical cultures and analyzed neuronal death twenty-four hours following administration of NMDA (a model of NMDAR excitotoxicity) or oxygen-glucose deprivation (a model of ischemic injury). In cultures prepared from wild-type mice, over-expression and knockdown of Cx36-containing gap junctions augmented and prevented, respectively, neuronal death from NMDAR-mediated excitotoxicity and ischemia. In cultures obtained form from Cx36 knockout mice, re-expression of functional gap junction channels, containing either neuronal Cx36 or non-neuronal Cx43 or Cx31, resulted in increased neuronal death following insult. In contrast, the expression of communication-deficient gap junctions (containing mutated connexins) did not have this effect. Finally, the absence of ethidium bromide uptake in non-transduced wild-type neurons two hours following NMDAR excitotoxicity or ischemia suggested the absence of active endogenous hemichannels in those neurons. Taken together, these results suggest a role for neuronal gap junctions in cell death via a connexin type-independent mechanism that likely relies on channel activities of gap junctional complexes among neurons. A possible contribution of gap junction channel-permeable death signals in neuronal death is discussed.
药理学阻断或基因敲除含神经元连接蛋白36（Cx36）的缝隙连接可减少由缺血、创伤性脑损伤和NMDA受体（NMDAR）介导的兴奋性毒性所导致的神经元死亡。然而，Cx36缝隙连接是通过通道依赖还是非通道依赖机制导致神经元死亡仍是一个悬而未决的问题。为了解决这个问题，我们通过慢病毒转导小鼠神经元皮质培养物来操纵连接蛋白的表达，并在给予NMDA（NMDAR兴奋性毒性模型）或氧糖剥夺（缺血性损伤模型）24小时后分析神经元死亡情况。在野生型小鼠制备的培养物中，含Cx36的缝隙连接的过表达和敲低分别增强和预防了NMDAR介导的兴奋性毒性和缺血导致的神经元死亡。在从Cx36基因敲除小鼠获得的培养物中，功能性缝隙连接通道的重新表达，无论是含神经元Cx36还是非神经元Cx43或Cx31，都会导致损伤后神经元死亡增加。相比之下，缺乏通讯功能的缝隙连接（含突变连接蛋白）的表达则没有这种作用。最后，在NMDAR兴奋性毒性或缺血2小时后，未转导的野生型神经元中没有溴化乙锭摄取，这表明这些神经元中不存在活跃的内源性半通道。综上所述，这些结果表明神经元缝隙连接通过一种可能依赖于神经元间缝隙连接复合物通道活性的连接蛋白类型非依赖机制在细胞死亡中发挥作用。文中还讨论了缝隙连接通道可通透的死亡信号在神经元死亡中的可能作用。
